DNA assays for genetic discrimination of three Phragmites australis subspecies in the United States

Abstract Premise To genetically discriminate subspecies of the common reed (Phragmites australis), we developed real‐time quantitative (qPCR) assays for identifying P. australis subsp. americanus, P. australis subsp. australis, and P. australis subsp. berlandieri. Methods and Results Utilizing study‐generated chloroplast DNA sequences, we developed three novel qPCR assays. Assays were verified on individuals of each subspecies and against two non‐target species, Arundo donax and Phalaris arundinacea. One assay amplifies only P. australis subsp. americanus, one amplifies P. australis subsp. australis and/or P. australis subsp. berlandieri, and one amplifies P. australis subsp. americanus and/or P. australis subsp. australis. This protocol enhances currently available rapid identification methods by providing genetic discrimination of all three subspecies. Conclusions The newly developed assays were validated using P. australis samples from across the United States. Application of these assays outside of this geographic range should be preceded by additional testing.

Convergent evolution, ecophenotypic variation, age-related morphological changes, hybridization, and the occurrence of cryptic species are some of the potentially confounding factors that make identifying some plants morphologically with absolute confidence difficult. This is especially true when populations include native and introduced lineages of the same species or subspecies (Meyerson et al., 2012;Gabby, 2020). The common reed, Phragmites australis (Cav.) Trin. ex Steud., is a perennial grass found in wetlands, marshes, river edges, lake shores, ponds, and roadside ditches across North America, tolerating fresh to mesohaline salinities . Three subspecies of P. australis co-occur in North America (Saltonstall, 2016): one native (P. australis subsp. americanus Saltonst., P. M. Peterson & Soreng), one introduced (P. australis subsp. australis), and one with uncertain origins (P. australis subsp. berlandieri (E. Fourn.) Saltonst. & Hauber).
Phragmites australis subsp. americanus is widespread across the United States, excluding the southeastern states (Saltonstall et al., 2004). Phragmites australis subsp. australis is of European origin, with multiple introductions leading to its spread across North America during the past couple of centuries (Saltonstall, 2002;Kirk et al., 2011); it is now recorded in every U.S. state (Saltonstall et al., 2004). Phragmites australis subsp. berlandieri is found along the U.S. Gulf Coast (Saltonstall, 2016). While cryptogenic in the United States (Lambert et al., 2016), with many populations being considered hybrids of P. australis subsp. berlandieri and P. mauritianus Kunth Lambert et al., 2016) or P. karka (Retz.) Trin. ex Steud. (Saltonstall and Hauber, 2007), it is not considered invasive in most areas . Commonly referred to as the Gulf Coast common reed, it is not endemic to the United States, but is native to Mexico, where multiple new haplotypes have been discovered (Colin and Eguiarte, 2016), and likely spread northward into the United States during the Pleistocene (Colin and Eguiarte, 2016). Both the introduced and Gulf Coast common reed are exhibiting range expansion, with more recent establishments in the U.S. Southwest, the only region worldwide that is inhabited by all three subspecies (Meyerson et al., 2010).
Native populations of common reed are ecologically important for maintaining high-quality wildlife habitat and species diversity, and are legally protected in many locations (Martin and Blossey, 2013); however, P. australis subsp. americanus is often outcompeted by the introduced subspecies, which grows more aggressively (Gabby, 2020) and can form dense monotypic stands (Swearingen and Saltonstall, 2010). Phragmites australis subsp. australis is managed by mowing, flooding, burning, and herbicide application because it alters wetland hydrology (increasing evaporation and trapping sediment) and degrades wildlife habitat, often reducing biodiversity (Chambers et al., 1999;Tewksbury et al., 2002). In many cases, genetic discrimination between the native and introduced common reed is important to natural resource managers interested in controlling invasions of the exotic subspecies while conserving populations of the native subspecies.
Although negative ecological impacts have been documented across the United States as resulting from the introduced subspecies, both P. australis subsp. australis and P. australis subsp. berlandieri provide important ecological services along the northern coast of the Gulf of Mexico. Their thick networks of roots/rhizomes provide storm surge protection and reduce erosion of coastal lands (Meyerson et al., 2009;Saleh and Weinstein, 2016). Whereas these subspecies thrive in conditions on the northern Gulf Coast (e.g., high salinity and moderate water depth), the native subspecies is absent (Saltonstall et al., 2005). However, Gulf Coast Phragmites stands are being negatively impacted by a nonindigenous scale insect (Nipponaclerda biwakoensis), with apparent differences in damage levels among Phragmites lineages in the region (Knight et al., 2018;Cronin et al., 2020). Chromosome ploidy level may also play a role in plant response to the scale insect (Meyerson et al., 2020), as P. australis subsp. australis is tetraploid and P. australis subsp. berlandieri is both tetraploid and hexaploid (Meyerson et al., 2016). Discriminating among subspecies thus becomes particularly important to coastal vegetation management, erosion, and flood control in the region. Additionally, in other regions of the United States, subspecies discrimination may be key to effective biocontrol of the introduced common reed (Tewksbury et al., 2002). As such, all three subspecies are mapped and managed throughout much of the United States and Canada.
To assist with mapping and management, and to streamline the genetic discrimination of P. australis, we created a tool capable of quickly identifying all three subspecies present in the United States by designing a set of novel DNA assays. The DNA assays developed here are realtime quantitative PCR (qPCR) assays targeting DNA loci unique to a particular taxon, such that they may be used to clearly identify specimens of that taxon. DNA assays have been shown to be a reliable tool to discriminate between morphologically similar plants (Duminil and Michele, 2009). The assays described in this study are hydrolysis probe-based qPCRs, capable of discriminating across very small differences in DNA sequence (1-3 base pairs) in the target loci. These assays are highly effective and provide more rapid differentiation of unidentified samples than other currently available Phragmites genetic discrimination methods (e.g., sequencing, Saltonstall, 2002;genotyping, Saltonstall, 2003;RNase H-dependent PCR, Zuzak et al., 2018) and can differentiate all three subspecies, unlike some Phragmites genetic discrimination methods (e.g., restriction fragment length polymorphism [PCR-RFLP], Wendell et al., 2021). Our hope is that further work and technological advances will allow these assays to be easily utilized for in-field plant identification.

METHODS AND RESULTS
Plant tissue samples (leaves) were collected from individual P. australis plants (n = 40) maintained at Louisiana State University, Baton Rouge, Louisiana, and included individuals representative of the different subspecies and lineages found across the United States. Additional samples were obtained from a U.S. Geological Survey greenhouse maintained at the Great Lakes Science Center in Ann Arbor, Michigan (n = 8); a University of Rhode Island greenhouse in Kingston, Rhode Island (n = 7); and field collections from Ohio (n = 1) and New York (n = 3) (Appendix 1). Leaf tissue samples from two morphologically similar, non-target confamilial species that co-occur with Phragmites in the study region, Arundo donax L. (n = 4) and Phalaris arundinacea L. (n = 4), were also obtained from the field for cross-amplification tests.
DNA extractions using a modified cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle, 1987;Lalhmangaihi et al., 2015) were performed for all samples (N = 67; Appendix 1) after an initial disruption step with a bead beater and Lysing Matrix D tubes (MP Biomedicals, Irvine, California, USA). Chloroplast DNA extractions were performed for nine of those samples, including all three subspecies and five biotypes (Table 1), following the Mariac et al. (2000) protocol. Genomic DNA concentrations (ng/µL) were determined with the NanoDrop One/One UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, Delaware, USA). DNA used for sequencing and primer testing had purity values (260/280 ratio) ranging from 1.77-1.98, and all samples were normalized to 1 ng/µL for qPCR assays. Native (P. australis subsp. americanus: n = 4), introduced (P. australis subsp. australis: n = 4), and Gulf Coast (P. australis subsp. berlandieri: n = 1) samples representing all U.S. lineages, and as many recognized "biotypes" within our study regions as were available in our sample pool, were selected for chloroplast sequencing (Table 1; Lambertini et al., 2012;Saltonstall, 2016). Each sample was prepared according to the protocol outlined for the Nextera DNA Flex Library Prep Kits (Illumina, San Diego, California, USA) for chloroplast whole genome sequencing. Sequencing was performed on the Illumina MiSeq system (Illumina) using the 600-cycle MiSeq Reagent V3 kit (Illumina). MiSeq Reporter Software (Illumina) was used to sort the pool of sequences by indices to identify the sequences from each sample. Chloroplast DNA genomes were assembled by aligning the reads of a reference sequence from the National Center for Biotechnology Information (NCBI) GenBank genetic sequence database (KJ825856.1) using the medium sensitivity/fast settings in Geneious Prime software (Biomatters Ltd., Auckland, New Zealand). Consensus sequences from each sample's whole chloroplast DNA were extracted and then aligned using global alignment with the free end gaps type function in Geneious Prime 2019.2.3. The entire chloroplast sequences (>137,500 bp) for each of the nine samples (Table 1) were submitted to the NCBI GenBank genetic sequence database; assigned accession numbers included: P. australis subsp. americanus (MW287631, MW287632, MW287634, MW287635), P. australis subsp. australis (MW287627, MW287628, MW287630, MW287633), and P. australis subsp. berlandieri (MW287629).
These chloroplast sequences were then used to identify multiple DNA loci with putative subspecies-unique sequence, from which nine qPCR assays (e.g., forward and reverse primers, and internal hydrolysis probe) were designed using Geneious and Primer3 (Rozen and Skaletsky, 2000). These assays were tested for expected detection/non-detection (i.e., detectable PCR amplification) in the nine original Phragmites samples used for chloroplast genome sequencing (chloroplast DNA extractions), of which three assays (one amplifying samples from P. australis subsp. americanus [AMER], one amplifying samples of P. australis subsp. australis and/or P. australis subsp. berlandieri [AUBE], and one amplifying samples of P. australis subsp. americanus and/or P. australis subsp. australis [AMAU]) ( Table 2) performed as expected and went through further optimization. Optimization of the three assays included minor adjustments to annealing temperature and cycle number in order to maximize amplification while maintaining non-amplification of nontarget species/subspecies. These three optimized assays were then validated using CTAB DNA extractions in all collected samples from each of the three subspecies (P. australis subsp. americanus: n = 24, P. australis subsp. australis: n = 26, and P. australis subsp. berlandieri: n = 9, including n = 9 "blind" samples for which only our collectors knew the morphological identity). The assays were then verified against four samples from each of the two non-target species, A. donax and P. arundinacea. We determined detection/non-detection results and threshold cycle (Ct) values over three technical replicates of each sample.
All qPCR reactions were run in 20 µL volumes containing 1X TaqMan Environmental Master Mix 2.0 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.5 µM of each primer, 0.125 µM of each probe, and 1 µL of DNA template (1 ng/µL). Temperature cycling began with an initial denaturation step at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. qPCR reactions were run on an Applied Biosystems ViiA 7 Real-Time PCR System (Thermo Fisher Scientific).
Three qPCR "technical replicates" were averaged to obtain mean Ct values for each sample across each assay (Appendix 1). The limit of detection (LOD) and limit of quantification (LOQ) for each assay were evaluated using qPCRs across several DNA template concentration levels using synthetic gBlocks Gene Fragments (Integrated DNA T A B L E 1 Chloroplast sequence sample information, including region of the United States, lineage, Phragmites australis subspecies, biotype as described in Lambertini et al. (2012) and Saltonstall (2016), collection state and location (latitude and longitude), and NCBI GenBank accession number. In our assay trials, Ct values from triplicate qPCR replicates were averaged for each sample (Appendix 1), then all sample Ct values were averaged by subspecies in order to characterize sensitivity (Figure 1). Assay sensitivity was also demonstrated by determining the LOD and LOQ for each assay (Table 3). LOD for AMER and AMAU were calculated from 24 replicates of four concentrations: 16, 8, four, and two copies/reaction. LOD for AUBE was calculated from 24 replicates of six concentrations: 2.5 million, 2 million, 1.25 million, 750,000, 500,000, and 375,000 copies/reaction. LODs for the three assays were 16 copies/reaction for AMER, eight copies/reaction for AMAU, and 2 million copies/reaction for AUBE. LOQ for AMER and AMAU were calculated from eight replicates of eight T A B L E 2 Chloroplast DNA assays, including qPCR primers and probes, designed and tested for Phragmites australis subspecies, listing assay name, primer or probe designation, primer or probe sequence, amplicon length, gene of origin, and targeted subspecies.

AUBE-R TCGCCATATCCCCATTTTCCTT
F I G U R E 1 Performance of three novel qPCR assays for discriminating among Phragmites australis subspecies as determined by average threshold cycle (Ct) values (± standard deviation) across three technical replicates per sample (n = 24 for P. australis subsp. americanus, n = 26 for P. australis subsp. australis, n = 9 for P. australis subsp. berlandieri, n = 4 for Arundo donax, n = 4 for Phalaris arundinacea), with DNA normalized to 1 ng/µL for each qPCR.
Lower Ct values indicate superior assay efficiency and greater likelihood of detection when DNA concentration is low. Species and subspecies for which no Ct value bar is shown did not amplify with that marker (e.g., no detection); these non-target species/subspecies do not have a Ct value of "0".
concentrations: 128, 64, 32, 16, eight, four, two, and one copies/reaction. LOQ for AUBE was calculated from eight replicates of eight concentrations: 20 million, 10 million, 5 million, 2.5 million, 2 million, 1.25 million, 750,000, and 500,000 copies/reaction. LOQs for AMER and AMAU were 128 copies/reaction and 64 copies/reaction, respectively. LOQ for AUBE was not determined, as the coefficient of variation (CV) for different concentration classes not only ranged above 35% for all classes, but also did not demonstrate the expected pattern of declining CV (i.e., increasing precision) with increasing template concentration (Klymus et al., 2020). Given the lack of that expected pattern, and a testing range that was already in the millions of copies (and thus of little practical value), further testing to determine an LOQ for AUBE was deemed unnecessary.
No amplification was observed in any DNA extraction blanks or negative template controls for any assay.

CONCLUSIONS
Three probe-based qPCR assays were designed to genetically distinguish between three P. australis subspecies. One assay (AMER) amplifies only samples from P. australis subsp. americanus, another assay (AUBE) amplifies only samples of P. australis subsp. australis and/or P. australis subsp. berlandieri, and another assay (AMAU) amplifies only samples of P. australis subsp. americanus and/or P. australis subsp. australis. Three basic scenarios for use of these assays are described in Appendix 2. First, an unknown sample could be assayed with each assay separately until identification is reached. Second, where the introduced and Gulf Coast subspecies co-occur, or where all three subspecies co-occur, DNA from an individual plant could be concurrently assayed (multiplexed) with AMAU and AUBE. If the sample amplifies with both AMAU and AUBE, the sample is assigned to the introduced subspecies, P. australis subsp. australis. If the sample amplifies only with AUBE, it is assigned to the Gulf Coast subspecies, P. australis subsp. berlandieri. If the sample amplifies only with AMAU, it is assigned to the native subspecies, P. australis subsp. americanus. Where only the native and introduced subspecies co-occur, DNA from an individual plant could be run with only the AMER assay. If the sample amplifies with AMER, it is assigned to the native subspecies, P. australis subsp. americanus, and if it does not amplify, it is assigned to the introduced subspecies, P. australis subsp. australis. The sample could also be multiplexed with AMER and AUBE, where amplification with AUBE (and not AMER) would demonstrate that the non-amplification with AMER was not due to technical failure (e.g., DNA extraction, pipetting, or qPCR instrument error), and the sample would then be assigned with additional confidence to the introduced subspecies, P. australis subsp. australis. Because qPCR assays often perform with less efficiency (and higher LOD) when multiplexed, changes to sensitivity when multiplexing should be assessed, particularly for low-quality samples, prior to assaying full sample sets. However, these assays were designed for the genetic discrimination of leaf tissue samples, for which the concentrations of template should, in nearly any case, be orders of magnitude above those at which efficiency and LOD would influence the outcomes. All else being equal, including DNA template concentrations, lower Ct values indicate comparatively greater assay efficiency and greater likelihood of providing DNA detection when DNA is extremely dilute or degraded. We suspect that the performance of AUBE, in terms of sensitivity and precision (i.e., high LOD, indeterminable LOQ at high concentrations), is due to a one base pair 5ʹ-3ʹ overlap in the forward primer and probe-binding sequence. Even with the large amount of chloroplast DNA sequence made available by our sequencing efforts, finding regions of DNA with adequate reaches of conserved and diverged sequence among the subspecies proved very difficult. The targeted regions were deemed the best options for developing the discriminatory assays, despite the primer/ probe overlap and apparent interference in the AUBE assay. We note that while sensitivity and precision are critical components for qPCR assays that will be used with DNA sources that may be poor in quality and/or low in concentration (e.g., environmental DNA [eDNA]), the AMER, AMAU, and AUBE assays were not designed for such purposes. The assays instead are designed for simple genetic discrimination using DNA from DNA-rich samples (e.g., leaf tissue), with no need for quantitative estimates of DNA template concentrations. Even very small, degraded, or small and degraded pieces of plant tissues will nearly always contain adequate concentrations of DNA template for qPCR amplification (Doyle and Doyle, 1987) with any of the assays. In such cases where one of these assays appears to not work with a suspected Phragmites sample, increasing the amount of DNA template in a qPCR (if too little template is suspected due to poorly preserved tissue) or, if enzymatic inhibition is suspected, diluting the DNA aliquot T A B L E 3 Efficiency of three novel Phragmites australis subspeciesdiscriminating qPCR assays (AMER, AMAU, and AUBE), including the limit of detection (LOD) and limit of quantification (LOQ) for each, determined using synthetic gBlocks Gene Fragments (Integrated DNA Technologies) and sensitivity calculations following Klymus et al. (2020). added to a qPCR are well-known, easily implemented steps that often overcome such challenges.
In this study, we developed three novel qPCR assays to genetically discriminate morphologically similar subspecies of the common reed in the United States. These three P. australis assays are accurate (zero non-target amplification and consistent target amplification) and offer faster identification of unknown samples than existing methods of Phragmites genetic discrimination (e.g., sequencing, Saltonstall, 2002;genotyping, Saltonstall 2003; RNase H-dependent PCR, Zuzak et al., 2018) to the subspecies level among all three P. australis subspecies present in the United States (e.g., PCR-RFLP, Wendell et al., 2021). The specific advantages and disadvantages of each available genetic discrimination method are summarized in Table 1 of Lindsay et al. (2022). We expect these qPCR assays to be valid for the discrimination of P. australis subspecies across the United States, including regions of southern California and southern Arizona where all three subspecies co-occur, because our sample set included specimens from these regions (Appendix 1). We suspect the assays to be applicable to P. australis subspecies in Canada and Mexico as well, although additional validation using samples from regions outside the United States would be required before implementation outside the assay design region, along with testing of any morphologically similar species (aside from A. donax and P. arundinacea) that occur in the region of new application. This step is important to ensure that the assays perform similarly on conspecific samples that may have small but impactful sequence differences within assay loci (e.g., nucleotide changes in the 3′ region of a primer resulting in substantially diminished primer-template binding efficiency).
The three P. australis subspecies for which the assays were developed are of substantial ecological and infrastructure significance to the Great Lakes and Gulf Coast regions. Knowing the geographic extent of each subspecies in finer detail could lead to improved mapping capabilities and better information on the susceptibility of coastal Louisiana locales to damages from N. biwakoensis, making long-term and geographically broad tracking of changes in the ranges of native and invasive Phragmites more feasible. Potential uses of these genetic assays include identification of resistant subspecies for use in ecosystem management and restoration along the Gulf Coast and an increased understanding of the extent of non-native invasion and dynamics in the Great Lakes region. Initial attempts to employ these assays with field-portable, user-friendly technologies have proven challenging (unpublished data), but near-future developments in rapid, portable DNAcharacterizing (e.g., qPCR) technologies should allow these and other assays to be used in the field by non-geneticists. The emergence of these capabilities into field studies could substantially empower Phragmites research and management by permitting practically (if not literally) instantaneous association of a sample or stand to a particular Phragmites group.